Since no microbiological cause has been identified in a significant proportion of patients with respiratory tract infections (Macfarlane, J. T. et al., 1993, Prospective study of aetiology and outcome of adult lower-respiratory-tract infections in the community, Lancet 341:511-514; Ruiz, M., S. et al., 1990, Etiology of community-acquired pneumonia: impact of age, comorbidity, and severity, Am. J. Respir. Crit. Care Med. 160:397-405), research has been conducted to identify possible novel agent(s). Of the three novel agents identified in the recent three years, including human metapneumovirus (Van den Hoogen, et al., 2001, A newly discovered human pneumovirus isolated from young children with respiratory tract disease, Nat. Med. 7:719-724), Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) (Peiris, J. S. et al., 2003, Coronavirus as a possible cause of severe acute respiratory syndrome, Lancet 361:1319-1325) and human coronavirus NL63 (HCoV-NL63) (Fouchier, R. A. et al., 2004, A previously undescribed coronavirus associated with respiratory disease in humans, Proc. Natl. Acad. Sci. USA. 101:6212-6216; van der Hoek, et al., 2004, Identification of a new human coronavirus, Nat. Med. 10:368-373), two were coronaviruses. Coronaviruses possess the largest genome of about 30 kb among all RNA viruses. As a result of the unique mechanism of viral replication, coronaviruses have a high frequency of recombination.
Based on genotypic and serological characterization, coronaviruses were divided into three distinct groups, with human coronavirus 229E (HCoV-229E) being a group 1 coronavirus and HCoV-OC43 a group 2 coronavirus (Lai, M. M. et al., 1997, The molecular biology of coronaviruses, Adv. Virus Res. 48:1-100). They account for 5-30% of human respiratory tract infections. In late 2002 and 2003, the epidemic caused by SARS-CoV affected over 8000 people with 750 deaths (for example, Peiris, J. S. et al., 2003, Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study, Lancet 361:1767-1772). We have also reported the isolation of SARS-CoV-like viruses from Himalayan palm civets, which suggested that animals could be the reservoir for the ancestor of SARS-CoV (Guan, Y. et al., 2003, Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China, Science 302:276-278). On the basis of genome analysis, SARS-CoV belongs to a fourth group of coronavirus, or alternatively, a distant relative of group 2 coronaviruses (Eickmann, M. et al., 2003, Phylogeny of the SARS coronavirus, Science 302:1504-1505; Marra, M. A. et al., 2003, The Genome sequence of the SARS-associated coronavirus, Science 300:1399-1404; Rota, P. A. et al., 2003, Characterization of a novel coronavirus associated with severe acute respiratory syndrome, Science 300:1394-1399; Snijder, E. J. et al., 2003, Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage, J. Mol. Biol. 331:991-1004; Yeh, S. H. et al., 2004, Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: molecular epidemiology and genome evolution, Proc. Natl. Acad. Sci. USA. 101:2542-2547). Recently, a novel group 1 human coronavirus associated with respiratory tract infections, HCoV-NL63, has been discovered, and its genome sequenced (37).
In January, 2004, a 71-year-old Chinese man was admitted to hospital because of fever and chills for two days associated with sore throat, rhinorrhoea, productive cough with purulent sputum, headache and nausea. He had history of pulmonary tuberculosis more than 40 years ago complicated by cicatrization of right upper lobe and bronchiectasis with chronic Pseudomonas aeruginosa colonization of airways. He was a chronic smoker and also had chronic obstructive airway disease, hyperlipidemia, and asymptomatic abdominal aortic aneurysm. He had just returned from Shenzhen of China three days before admission. During his three-day trip to Shenzhen, he had no history of contact with or consumption of wild animals. On admission, his oral temperature was 37.6° C. Physical examination showed tracheal deviation to the right and inspiratory crackles over the anterior left lower zone. His haemoglobin level was 14.7 g/dL, total white cell count 12.1×109/L, with neutrophil 9.7×109/L, lymphocyte 1.6×109/L and monocyte 0.5×109/L, and plate count 303×109/L. His liver and renal function tests were within normal limits. Chest radiograph showed right upper lobe collapse and new patchy infiltrates over the left lower zone. Blood culture was performed. Empirical oral amoxicillin/clavulanate and azithromycin were commenced. Nasopharyngeal aspirates for direct antigen detection for respiratory viruses, RT-PCR for influenza A virus, human metapneumovirus and SARS-CoV, and viral cultures were negative. Sputum for bacterial culture only recovered P. aeruginosa. Sputum for mycobacterial culture was negative. Blood culture was negative. Paired sera for antibodies against Mycoplasma, Chlamydia, Legionella, and SARS-CoV did not show any rise in antibody titres. His fever subsided two days after admission. His cough improved and he was discharged after five days of hospitalization. Amoxicillin/clavulanate and azithromycin were continued for a total of seven days. The present inventors were the group involved in the investigation of this patient. All tests for identifying commonly recognized viruses and bacteria were negative in these patients. The etiologic agent responsible for this disease was not known until the complete genome of CoV-HKU1 from this patient by the present inventors as disclosed herein. Further studies disclosed herein have revealed that CoV-HKU1 is a human coronavirus and there are, at least, two (2) genotypes, A and B, within CoV-HKU1. The invention is useful in both clinical and scientific research applications.